Studies on replication of plasmid colE1 DNA have been continued. Initiation of replication from a specific region of the plasmid can be carried out by purified enzymes. DNA dependent RNA polymerase, DNA polymerase I and ribonuclease H are essential and sufficient for the initiation. A transcript by RNA polymerase is probably processed by ribonuclease H and used as a primer for DNA synthesis which is carried out by DNA polymerase I. The superhelical turns are released by DNA replication. DNA gyrase is required for further elongation of the DNA strand to impose negative superhelical turns.